This project aims to determine the molecular genetic bases of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol. Steroid 21-hydroxylase activity is deficient in more than 90% of patients, whereas 11-hydroxylase is affected in most of the remaining cases. Therefore, this project concentrates on the ways in which mutations in the genes encoding these enzymes result in CAH. To study the mechanisms by which 21-hydroxylase deficiency mutations are generated, the polymerase chain reaction will be used with gene-specific primers to detect de novo deletions of the CYP21 genes generated by unequal crossing over in meiosis during spermatogenesis. It will be determined if these deletions cluster in specific regions of the CYP21 genes, if men differ in their propensity to generate such deletions in sperm, and if the CYP21 genes are more likely to undergo such deletions than other genes that are tandemly duplicated, such as the CYP11B, globin or visual pigment genes. The frequency of de novo deletions of CYP21 in sperm will be compared with that of de novo gene conversions. The clinical severity of 21-hydroxylase deficiency in individuals who are homo- or hemizygous for the most common mutation causing this disorder, a single base change activating a cryptic splice site, will be correlated with the degree of recognition of the normal site in cultured cells. An efficient method for allele detection in 21-hydroxylase deficiency will be developed using a nick ligation assay (ligase chain reaction). Additional mutations in CYP11B1 and CYP11B2 causing 11beta-hydroxylase and corticosterone methyloxidase II deficiencies will be identified by amplifying gene segments using PCR and then sequencing uncloned amplified DNA. The functional effects of each missense mutation will be determined by site-directed mutagenesis of cDNA and expression in cultured cells. Transcriptional regulatory elements in CYP11B1 and (especially) CYP11B2 will be identified using sequential deletions of 5' flanking regions in reporter constructs, gel retardation and DNAse I footprinting assays.